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Use of Sera from Humans and Dolphins with Lacaziosis and Sera from Experimentally Infected Mice for Western Blot Analyses of Lacazia loboi Antigens


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Antibodies in the sera of patients with lacaziosis recognized an approximately 193-kDa antigen and other Lacazia loboi antigens. Paracoccidioides brasiliensis gp43 antigen was detected by all evaluated sera, but they failed to detect a protein with the same molecular mass in L. loboi extracts. This study is the first to examine the humoral response to L. loboi antigens by using multiple host sera.
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CLINICAL AND VACCINE IMMUNOLOGY, Jan. 2008, p. 164–167 Vol. 15, No. 1
1556-6811/08/$08.000 doi:10.1128/CVI.00201-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.
Use of Sera from Humans and Dolphins with Lacaziosis and Sera from
Experimentally Infected Mice for Western Blot Analyses of
Lacazia loboi Antigens
Leonel Mendoza,
* Andre´a F. F. Belone,
Raquel Vilela,
Manuela Rehtanz,
Gregory D. Bossart,
John S. Reif,
Patricia A. Fair,
Wendy N. Durden,
Judy St. Leger,
Luiz R. Travassos,
and Patricia S. Rosa
Biomedical Laboratory Diagnostics Program, Department of Microbiology and Molecular Genetics, Michigan State University, East Lansing,
Michigan 48824-1031
; Instituto Lauro de Souza Lima, Bauru, Sa˜o Paulo, Brazil
; New York University, Department of Basic Sciences,
Infectious Diseases, New York, New York 10010
; Harbor Branch Oceanographic Institution, Center for Coastal Research,
Marine Mammal Research and Conservation, Fort Pierce, Florida 34946
; Department of Environmental and
Radiological Health Sciences, Colorado State University, Fort Collins, Colorado 80523
; National Oceanic and
Atmospheric Administration/National Ocean Service/Center for Coastal Environmental Health and
Biomolecular Research, Charleston, South Carolina 29412
; Hubbs-SeaWorld Research Institute,
Orlando, Florida 32821-8043
; Department of Pathology, SeaWorld, San Diego,
California 92109
; and Department of Microbiology, Immunology and
Parasitology, Universidade Federal de Sa˜o Paulo, Sa˜o Paulo, Brazil
Received 14 May 2007/Returned for modification 5 August 2007/Accepted 16 October 2007
Antibodies in the sera of patients with lacaziosis recognized an 193-kDa antigen and other Lacazia loboi
antigens. Paracoccidioides brasiliensis gp43 antigen was detected by all evaluated sera, but they failed to detect
a protein with the same molecular mass in L. loboi extracts. This study is the first to examine the humoral
response to L. loboi antigens by using multiple host sera.
Lacazia loboi is an uncultivated fungal pathogen predomi-
nantly restricted to Latin America. Lacaziosis has been re-
corded among humans dwelling in this geographical area, tour-
ists visiting areas of endemicity (4, 10), and workers handling
infected dolphins (19, 24). Lacaziosis has also been diagnosed
in dolphins inhabiting the coastal areas of the United States (9,
12, 19), Brazil (16), and France (25). Using molecular meth-
ods, the phylogenetic features of L. loboi were only recently
deciphered (13, 18, 31). These studies placed L. loboi as a sister
group to Paracoccidioides brasiliensis and, in turn, linked to the
other dimorphic members of the Onygenales in the family
The clinical features of Lacazia loboi and P. brasiliensis in-
fections are different, but both fungi develop yeast cells, which
are difficult to differentiate microscopically, in the host’s in-
fected tissues (7). This resemblance was used by some inves-
tigators to classify this pathogen in the genus Paracoccidioides
(1, 6, 8). Thus, due to L. loboi’s inability to grow in culture,
immunological studies have traditionally been carried out with
antigens extracted from mycelial cultures of P. brasiliensis (15,
17, 21, 23–27, 30). The objective of our study was to charac-
terize the immunogenic proteins extracted from L. loboi yeast-
like cells in Western blotting analyses, using sera from humans
and dolphins with lacaziosis and sera from experimentally in-
fected mice.
Sera from four human patients with lacaziosis from Acre
State, Brazil, and from one Brazilian patient with proven para-
coccidioidomycosis and four sera from experimentally L. loboi-
infected mice were collected and stored at 80°C until use (2).
Sera from three bottlenose dolphins (Tursiops truncatus) in-
fected with L. loboi were evaluated: one wild dolphin infected
with L. loboi was captured in the Indian River Lagoon, FL, one
was a stranded Indian River Lagoon bottlenose dolphin, and
one was a captive bottlenose dolphin from SeaWorld in San
Diego, CA. In addition, two sera from apparently healthy hu-
mans, two sera from healthy dolphins, and two sera from
healthy laboratory mice were used as negative controls.
Lacazia loboi yeast-like cell extracts were obtained from the
footpads of a previously infected BALB/c mouse 6 months
after inoculation, as described by Belone et al. (2). Briefly, L.
loboi yeast-like cells were then centrifuged for 10 min at 3,000
rpm, and the supernatant was discarded. Cells were washed
twice with saline, and the pellet was suspended in 1 ml of
saline. Fungal cells were macerated to a powder with liquid
nitrogen inside a mortar and used in the experiments. The
gp43 protein of P. brasiliensis was extracted and purified per
the method of Taborda et al. (28). Sodium dodecyl sulfate-
polyacrylamide gel electrophoresis and Western blot analyses
were done per the methods of Laemmli (14) and Towbin et al.
* Corresponding author. Mailing address: Biomedical Laboratory
Diagnostics Program, Michigan State University, 322 North Kedzie
Hall, East Lansing, MI 48824-1031. Phone: (517) 353-7800. Fax: (517)
432-2006. E-mail:
Published ahead of print on 24 October 2007.
The diagnosis of lacaziosis in the investigated humans and
dolphins was confirmed by clinical and histopathological meth-
ods (Fig. 1). The stained gels of L. loboi protein extracts
showed at least 10 bands, ranging from 193 kDa to 9.0 kDa
(Fig. 2). The L. loboi protein extract lacked a visible protein
band at 43 kDa. The purified gp43 protein of P. brasiliensis
showed a strong band at the expected molecular mass and did
not contain other proteins (Fig. 2). Normal mouse skin tissue
samples processed as described above did not show visible
bands (data not shown).
The immunoglobulin G (IgG) antibodies in the sera of all
tested hosts with lacaziosis detected the gp43 protein of P.
brasiliensis fairly well (Fig. 3). The tested dolphin, human, and
experimentally infected mouse sera detected a major, immu-
nodominant, 193-kDa antigen in the L. loboi protein extract
(Fig. 3) and also reacted weakly with other immunogenic an-
tigens (Fig. 3). The experimentally infected mouse antibodies
mildly detected five other immunogens, including an 48-kDa
protein not detected by the other sera (Fig. 3, panel 2, lane b,
arrowhead). The IgG antibodies of the dolphins detected only
two high-molecular-mass antigens in the L. loboi protein ex-
tract, including the immunodominant, 193-kDa antigen (Fig.
3, panel 3, lane b). The antibodies in the serum from a patient
with paracoccidioidomycosis weakly detected at least 13 other
antigenic components in the L. loboi protein extract, ranging
from 193 kDa to 9.0 kDa. Some of these antigens were not
detected by the antibodies in the sera from humans and dol-
phins with lacaziosis or the antibodies in the sera from exper-
imentally infected mice (Fig. 3, panel 4, lane b). Only the
antibodies in the serum from the patient with paracoccidioid-
omycosis weakly detected a molecular antigen of 43 kDa in the
L. loboi protein extract (Fig. 3, panel 4, lane b). The healthy
FIG. 1. Human parakeloidal (A) and dolphin verrucous (B) lesions
from two of the hosts with lacaziosis used in this study. The histopa-
thology of these lesions is shown in panels C (human) (silver stain;
magnification, 100) and D (dolphin) (silver stain; magnification,
FIG. 2. Coomassie blue-stained sodium dodecyl sulfate-polyacryl-
amide gel showing purified Paracoccidioides brasiliensis gp43 (lane 1)
and the profile of an Lacazia loboi protein extract (lane 2). The figure
shows the 193-kDa immunodominant antigen (arrow) as well as
other proteins in the L. loboi protein extract. The asterisks indicate
common proteins detected by the sera tested in this study. Lane M,
molecular mass marker.
FIG. 3. Western blot analyses of transferred proteins on nylon
membranes. The antibodies in the serum from a human with lacaziosis
weakly detected Paracoccidioides brasiliensis gp43 (lane 1a). This se-
rum strongly detected an 193-kDa immunodominant antigen (arrow)
as well as six other weak bands (asterisks) (lane 1b). The IgG in the
serum from the experimentally infected mouse weakly detected P.
brasiliensis gp43 (lane 2a). The mouse antibodies strongly detected the
193-kDa immunodominant antigen (arrow) as well as seven other
proteins (asterisks) and an 48-kDa protein not detected by the other
sera (lane 2b, arrowhead). The antibodies in the dolphin with lacaziosis
slightly detected P. brasiliensis gp43 (lane 3a). The antibodies strongly
recognized the 193-kDa immunodominant antigen (arrow) and only
one of the minor antigens previously detected in the L. loboi extract
(lane 3b). The antibodies in the serum from the patient with paracoc-
cidioidomycosis strongly detected gp43 (lane 4a). The antibodies of
this patient also detected the immunodominant 193-kDa antigen
(arrow) in the L. loboi extract as well as other antigens (asterisks),
including a 43-kDa antigen (arrowhead) (lane 4b). The molecular mass
marker (lanes M) appears in each of the panels.
OL. 15, 2008 NOTES 165
control human, dolphin, and mouse sera did not react with the
antigens used in this study (data not shown). One of the two
control dolphin sera possessed antibodies against the 43-kDa
purified protein of P. brasiliensis and the major antigens of L.
loboi normally detected by the dolphins with lacaziosis. Puta-
tive residual normal mouse skin cells were not detected by the
tested sera (data not shown).
With the exception of three previous studies (1, 23, 30), in
the past 75 years L. loboi immunological research has been
carried out using antigens of P. brasiliensis (6, 8, 15, 17, 21, 24,
26, 27, 32). Our study showed that patients with lacaziosis
possessed antibodies not only against the gp43 antigen of P.
brasiliensis but mainly against an 193-kDa immunodominant
L. loboi antigen. The possibility that the 193-kDa antigen
comprises several compressed L. loboi immunogens is cur-
rently under investigation in our laboratory. The IgGs in the
sera from patients with lacaziosis failed to detect the gp43-like
antigen in the protein extract of L. loboi. However, the serum
from the patient with paracoccidioidomycosis did detect an
antigen with a similar molecular mass. The cross-reactive an-
tigens reported in this study, including purified gp43 and those
reported by other investigators (1, 6, 15, 21, 24, 26, 30), were
not unexpected, since recent molecular studies have shown
that both L. loboi and P. brasiliensis share the same ancestor
(13, 18, 31).
The many reports of lacaziosis in dolphins have puzzled
investigators for decades (3, 5, 9, 12, 19, 22, 25, 32). However,
studies to elucidate its etiology, epidemiology, and immunol-
ogy have been equally challenging (5, 22, 25). Our study
showed that dolphins develop IgG antibodies against similar
prominent antigens to those recognized by humans with laca-
ziosis and by experimentally infected mice. Interestingly, one
of the negative control dolphins detected the gp43 antigen of
P. brasiliensis and the other major antigens of L. loboi identi-
fied by dolphins with lacaziosis. This unexpected result sug-
gests a previous exposure to L. loboi without disease, as it is
frequent among the other dimorphic members of the Onyge-
nales (15, 17, 20). Thus, Western blotting analyses could be a
useful tool for epidemiological studies of L. loboi. Although
some investigators have reported morphological differences
between the yeast-like cells of L. loboi in both species (11), our
study is the first to suggest that dolphins are infected with
similar L. loboi strains to those infecting humans.
In contrast to previous serological reports (1, 23, 30), this
study suggests that during infection, L. loboi presents immu-
nogens to the immune system which are different from that in
P. brasiliensis infection. The molecular characterization of
these antigens, especially the 193-kDa immunodominant an-
tigen, could generate valuable information to better under-
stand the immunology and serology of L. loboi infection and
possibly aid in the development of new therapies for infections
caused by this resilient fungal pathogen.
Lesions from the free-ranging dolphin were collected under Na-
tional Marine Fisheries Service scientific research permit no. 998-1678,
issued to Gregory D. Bossart as part of the Bottlenose Dolphin Health
and Risk Assessment (HERA) Project conducted in the Indian River
Lagoon, FL, and the estuarine waters of Charleston, SC. Samples from
the stranded dolphin were collected under a National Marine Fisheries
Service letter of agreement, and this collection was supported in part
under award NA06NMF4390138 from the National Oceanic and At-
mospheric Administration, U.S. Department of Commerce. This re-
search was supported in part by the Biomedical Laboratory Diagnosis
Program, Michigan State University, MI.
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... Studies using serum samples from humans and dolphins with lacaziosis/lobomycosis, mice experimentally infected with L. loboi, and serum samples from humans with paracocidioidomycosis showed that IgG in serum samples from dolphins and humans infected with L. loboi had strong cross-reactivity with the gp43 antigen of P. brasiliensis (4,6,21). These findings support the hypothesis that the uncultivated organism causing cutaneous granulomas in humans and dolphins was L. loboi. ...
... Findings also implied that the gp43 antigen of the etiologic agent of parakeloidallike granulomas in humans and dolphins was antigenically similar to that of P. brasiliensis. On the basis of these serologic studies (4,6,21), current phylogenetic data for gp43 and Kex gene exons, and ITS DNA sequences, placement of L. loboi from humans in its own genus is questionable. Efforts to culture the organism from dolphins on classical laboratory media successfully used to isolate P. brasiliensis from humans with paracoccidioidomycosis were not successful (4,7). ...
Full-text available
2097 Cutaneous granulomas in dolphins were believed to be caused by Lacazia loboi, which also causes a similar disease in humans. This hypothesis was recently challenged by reports that fungal DNA sequences from dolphins grouped this pathogen with Paracoccidioides brasiliensis. We conducted phylogenetic analysis of fungi from 6 bottle-nose dolphins (Tursiops truncatus) with cutaneous granu-lomas and chains of yeast cells in infected tissues. Kex gene sequences of P. brasiliensis from dolphins showed 100% homology with sequences from cultivated P. brasil-iensis, 73% with those of L. loboi, and 93% with those of P. lutzii. Parsimony analysis placed DNA sequences from dolphins within a cluster with human P. brasiliensis strains. This cluster was the sister taxon to P. lutzii and L. loboi. Our molecular data support previous findings and suggest that a novel uncultivated strain of P. brasiliensis restricted to cutaneous lesions in dolphins is probably the cause of lacaziosis/lobomycosis, herein referred to as paracoccidi-oidomycosis ceti.
... No serological tests are currently available for commercial use.Western blot analysis and PCR techniques have been developed. 110 A fluorescein diacetateethidium bromide (FDEB) staining technique has been found to be reliably sensitive and specific in determining viability of L. loboi to measure therapeutic success. 110,111 Treatment Wide surgical excision ensuring that margins are free of infection, remains the most successful treatment. ...
... 110 A fluorescein diacetateethidium bromide (FDEB) staining technique has been found to be reliably sensitive and specific in determining viability of L. loboi to measure therapeutic success. 110,111 Treatment Wide surgical excision ensuring that margins are free of infection, remains the most successful treatment. Electrodessication or cryosurgery is useful for early small lesions. ...
Full-text available
... They demonstrated that an immunodominant antigen with a high molecular weight of ∼193 kDa was recognized by antibodies in the serum, suggesting that the antigenic proteins of L. loboi have much higher molecular weight than the counterpart, gp43, of P. brasiliensis. 47 Preserved cellular immunity is probably necessary to hinder the progression of the disease, or in some cases, prevent its appearance. Nowadays it is not possible to identify sub-clinical lobomycosis due to lack of a specific and reliable antigen. ...
... 57 In contrast, serum from an infected dolphin recognized an immunodominant 193-kDa antigen from an extract of human L. loboi more strongly than the gp43 antigen of P. brasiliensis in western blotting analyses. 47 Experimental inoculation of a laboratory scientist with yeast-like cells from a human patient, as well as a report of an accidental transmission of lobomycosis in another laboratory scientist who collected and purified fungal cells from human skin biopsies, 50 imply that under unusual circumstances, Lacazia of human origin can be transmitted to other humans. 24 The epidemiologic data of lobomycosis in endemic areas is unknown. ...
Full-text available
Lobomycosis is a subcutaneous mycosis of chronic evolution caused by the Lacazia loboi fungus. Its distribution is almost exclusive in the Americas, and it has a particularly high prevalence in the Amazon basin. Cases of lobomycosis have been reported only in dolphins and humans. Its prevalence is higher among men who are active in the forest, such as rubber tappers, bushmen, miners, and Indian men. It is recognized that the traumatic implantation of the fungus on the skin is the route by which humans acquire this infection. The lesions affect mainly exposed areas such as the auricles and upper and lower limbs and are typically presented as keloid-like lesions. Currently, surgical removal is the therapeutic procedure of choice in initial cases. Despite the existing data and studies to date, the active immune mechanisms in this infection and its involvement in the control or development of lacaziosis have not been fully clarified. In recent years, little progress has been made in the appraisal of the epidemiologic aspects of the disease. So far, we have neither a population-based study nor any evaluation directed to the forest workers.
... One study demonstrated that individuals with lacaziosis possess antibodies reactive to the gp43 antigen of P. brasiliensis, and also to a 193 kDa major L. loboi antigen through WB. The cross-reactivity occurs because, as supported by molecular studies, L. loboi and P. brasiliensis share a similar ancestor [187]. In contrast to prior reports, this study proposes that, during infection, L. loboi presents antigens that are distinct from that presented during paracoccidioidomycosis [188,189]. ...
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The endemic mycoses blastomycosis, coccidioidomycosis, histoplasmosis, paracoccidioidomycosis, cryptococcosis, sporotrichosis, talaromycosis, adiaspiromycosis, and emergomycosis are mostly caused by geographically limited thermally dimorphic fungi (except for cryptococcosis), and their diagnoses can be challenging. Usual laboratory methods involved in endemic mycoses diagnosis include microscopic examination and culture of biological samples; however, serologic, histopathologic, and molecular techniques have been implemented in the last few years for the diagnosis of these mycoses since the recovery and identification of their etiologic agents is time-consuming and lacks in sensitivity. In this review, we focus on the immunologic diagnostic methods related to antibody and antigen detection since their evidence is presumptive diagnosis, and in some mycoses, such as cryptococcosis, it is definitive diagnosis.
... The amplicons were purified and then sequenced in both directions with the same primers using BigDye terminator chemistry in an ABI Prism 310 genetic analyzer (Perkin-Elmer Foster City, Cal.).Haplotype and principal component analyses. Based on the results of previous Paracoccidioides species genetic and serological analyses, the Gp43 and ADP-rf DNA coding regions were found to be useful in population and phylogenetic studies12,[14][15][16]30,52 . Thus, we selected their DNA sequence in these analyses. ...
Full-text available
Ever since the uncultivated South American fungal pathogen Lacazia loboi was first described 90 years ago, its etiology and evolutionary traits have been at the center of endless controversies. This pathogen infects the skin of humans and as long believed, dolphin skin. However, recent DNA analyses of infected dolphins placed its DNA sequences within Paracoccidioides species. This came as a surprise and suggested the human and dolphin pathogens may be different species. In this study, population genetic analyses of DNA from four infected dolphins grouped this pathogen in a monophyletic cluster sister to P. americana and to the other Paracoccidioides species. Based on the results we have emended the taxonomy of the dolphin pathogen as Paracoccidioides cetii and P. loboi the one infecting human. Our data warn that phylogenetic analysis of available taxa without the inclusion of unusual members may provide incomplete information for the accurate classification of anomalous species.
... As anticipated, the cells were morphologically similar in size and shape to previous descriptions of L. loboi isolated from dolphins. 5,11 We also utilized culture conditions under which P. brasiliensis is grown in vitro due to the similarities in molecular phylogeny, 2 serological cross-reactivity in Western blot analyses, 14 and geographic distribution between the organisms. Inoculation of Sabouraud Dextrose Agar and Blood Agar at 37 • C will elicit colony growth for P. brasiliensis. ...
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Lobomycosis (lacaziosis) is a chronic, granulomatous, fungal infection of the skin and subcutaneous tissues of humans and dolphins. To date, the causative agent, the yeast-like organism Lacazia loboi, has not been grown in the laboratory, and there have been no recent reports describing attempts to culture the organism. As a result, studies on the efficacy of therapeutics and potential environmental reservoirs have not been conducted. Therefore, the objective of the current study was to utilize both classical and novel microbiological methods in order to stimulate growth of Lacazia cells collected from dolphin lesions. This included the experimental inoculation of novel media, cell culture, and the use of artificial skin matrices. Although unsuccessful, the methods and results of this study provide important insight into new approaches that could be utilized in future investigations of this elusive organism.
... In humans, lobomycosis acts as a chronic fungal skin infection that mainly occurs in rural areas of South and Central America, where this disease is endemic (Brito and Quaresma 2007 ). According to Haubold et al. ( 2000 ) the etiologic agents responsible for lobomycosis in humans and dolphins are not the same; notwithstanding, evidence from serologic data suggests that humans and dolphins are infected by the same L. loboi strains (Mendoza et al. 2008 ). In dolphins, lobomycosis is distributed from southern Brazil to the Gulf of Mexico and Atlantic coast of Florida (Reif et al. 2006 ). ...
Historically, human activities have impacted cetacean populations; such activities have included commercial fishing operations, overexploitation of marine species, habitat degradation, and commercial harvesting. As a result, many species are now classified as being threatened (Gulland and Hall 2007; Sherman 2000). Additionally, most species are exposed to anthropogenic contaminants, even those inhabiting areas far from human activities (Aono et al. 1997; Bard 1999). Such exposure may lead to health problems, such as impairment of immunological resistance, increased susceptibility to infectious diseases, and reduced reproductive fitness (Ross and Birnbaum 2003; Tanabe 2002).
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Infections caused by the fungal pathogen Lacazia loboi were first reported in 1931 by Jorge de Oliveira Lobo in a human with granulomatous skin lesions in Pernambuco, Brazil. Early histopathological and serological analyses found morphological similarities and cross-reactive antigens with Paracoccidioides brasiliensis. In 1971, veterinarians working with dolphins in Florida, USA, reported granulomatous skin lesions in a dolphin, similar to that in human lacaziosis. Based on histopathological findings, L. loboi was initially believed to be also the etiologic agent of cutaneous disease in dolphins. Ever since, cutaneous granulomas have been reported in different dolphin species around the coast of Asia, Europe, and North and South America. Recently, using molecular biology approaches, some investigators stated that the DNA sequences extracted from cases of cutaneous granulomas in dolphins were closely related to those of P. brasiliensis. This chapter deals with the history, taxonomy, and other features of L. loboi in humans and the unculturable P. brasiliensis var. ceti type affecting the skin of dolphins.
There are no studies investigating the role of nutritional status and immunity associated with Jorge Lobo's disease. The objective of this study was to evaluate the effects of protein-calorie malnutrition on the immune response of BALB/c mice inoculated with Lacazia loboi. In this study,the animals were divided into four groups: G1: inoculated with restricted diet, G2: not inoculated with restricted diet, G3: inoculated with regular diet, G4: not inoculated with regular diet. The animals of groups G1 and G2 were submitted to malnutrition for 20 days and once installed the animals were inoculated intradermally into the footpad. After 4 months, they were euthanised for the isolation of peritoneal lavage cells and removal of the footpad. The production of IL-2, IL-4, IL-10, IL-12, IFN-γ, TNF-α, H2 O2 and nitric oxide (NO) was evaluated in the peritoneal lavage cells. The footpad was evaluated regarding the size of macroscopic lesions, number of fungi and viability index. The results showed that the infection did not exert great influence on the body weight of the mice and previous malnutrition was an unfavourable factor for viability index, number of fungi, macroscopic lesion size in the footpad and production of H2 O2 , NO, IL-12, IL-10 and IFN-γ, suggesting that malnutrition significantly altered fungal activity and peritoneal cells. The results suggest considerable interaction between nutrition and immunity in Jorge Lobo's disease. © 2015 Blackwell Verlag GmbH.
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We demonstrated through several immunochemical tests the presence of GP-43 from P. brasiliensis in extracts of cutaneous lesions from Jorge Lobo's disease. This glicoprotein is one of the immunodominant antigens in this species, and is used to identify it. The demonstration of GP-43 tissues infected by the agent of Jorge Lobo's disease is an additional evidence for classifying it in the genera Paracoccidioides, species loboiAtravés de várias provas imunoquímicas foi demonstrada a presença da GP-43 em extratos de lesões cutâneas da doença de Jorge Lobo. A glicoproteína de 43 kDa é um dos antígenos dominantes do Paracoccidioides brasiliensis, permitindo a identificação desta espécie fúngica. A demonstração da GP-43 em tecidos infectados com o agente da doença de Jorge Lobo, constitui mais um argumento para colocá-lo no gênero Paracoccidioides, espécie loboi
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Lacazia loboi is the last of the classical fungal pathogens to remain a taxonomic enigma, primarily because it has resisted cultivation and only causes cutaneous and subcutaneous infections in humans and dolphins in the New World tropics. To place it in the evolutionary tree of life, as has been done for the other enigmatic human pathogens Pneumocystis carinii andRhinosporidium seeberi, we amplified its 18S small-subunit ribosomal DNA (SSU rDNA) and 600 bp of its chitin synthase-2 gene. Our phylogenetic analysis indicated that L. loboi is the sister taxon of the human dimorphic fungal pathogen Paracoccidioides brasiliensis and that both species belong with the other dimorphic fungal pathogens in the order Onygenales. The low nucleotide variation among three P. brasiliensis 18S SSU rDNA sequences contrasts with the surprising amount of nucleotide differences between the two sequences of L. loboi used in this study, suggesting that the nucleic acid epidemiology of this hydrophilic pathogen will be rewarding.
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A method has been devised for the electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets. The method results in quantitative transfer of ribosomal proteins from gels containing urea. For sodium dodecyl sulfate gels, the original band pattern was obtained with no loss of resolution, but the transfer was not quantitative. The method allows detection of proteins by autoradiography and is simpler than conventional procedures. The immobilized proteins were detectable by immunological procedures. All additional binding capacity on the nitrocellulose was blocked with excess protein; then a specific antibody was bound and, finally, a second antibody directed against the first antibody. The second antibody was either radioactively labeled or conjugated to fluorescein or to peroxidase. The specific protein was then detected by either autoradiography, under UV light, or by the peroxidase reaction product, respectively. In the latter case, as little as 100 pg of protein was clearly detectable. It is anticipated that the procedure will be applicable to analysis of a wide variety of proteins with specific reactions or ligands.
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Sera from patients with paracoccidioidomycosis (PCM), histoplasmosis (HP), or Jorge Lobo's disease (JL) were titrated against purified gp43 from Paracoccidioides brasiliensis by using both enzyme-linked immunosorbent assay (ELISA) and immunoprecipitation (IPP) reactions with 125I-labeled antigens. In IPP, PCM sera and other sera could be distinguished on the basis of serum titers, whereas in ELISA, 53% of the HP sera and 29% of the JL sera reacted similarly to the PCM sera. To investigate the possible role of the carbohydrate epitopes in these reactions, we compared the reactivities of sera from several patients with native and deglycosylated gp43. Competition experiments were carried out with monosaccharides as inhibitors. The results suggest that greater than 85% of the reactions of the PCM sera with gp43 involved peptide epitopes. Cross-reactions with HP and JL sera in ELISA were predominantly attributed to periodate-sensitive carbohydrate epitopes containing galactosyl residues. HP and JL sera which reacted strongly with gp43 in ELISA were only weakly reactive or did not react in IPP with labeled antigens in solution. Moreover, ELISA reactions could be significantly inhibited either by monosaccharides or by periodate treatment. Apparently, carbohydrate epitopes in gp43 are more accessible to the antibodies when the molecule is bound to a plastic substrate than when it is in solution. Structural changes in the gp43 antigen arising by N deglycosylation abolish reactivity with PCM sera and support the existence of conformational peptide epitopes.
The fluorescent antibody technique was used to study antigenic relationships betweenParacoccidioides loboi and other pathogenic fungi. The findings suggest thatP. loboi is more closely related antigenically to certainP. brasiliensis strains than to others and that it has antigens in common with the yeast form ofHistoplasma capsulatum, H. duboisii, Blastomyces dermatitidis, Candida albicans and also the mycelial form ofCoccidioides immitis. Serum globulins from 3 cases of keloidal blastomycosis were labelled with fluorescein isothiocyanate. These conjugates showed slight or no reactivity withP. loboi, the yeast forms ofP. brasiliensis, H. capsulatum, H. duboisii andB. dermatitidis, However, they stained brightlyC. albicans, serotypes A and B, the tissue form ofC. immitis and the yeast form ofSporotrichum schenckii. Adsorption of these reagents withC. albicans eliminated all staining except that forS. schenckii. These patients had no history of clinical sporotrichosis.
A 73-year-old woman from Guyana had nodular keloidal lesions on her face which had persisted for 56 years. A biopsy of the lesion revealed lobomycosis, a rare fungal infection. Microscopically, numerous fungi were seen in giant cells and macrophages. The macrophages had abundant pink reticulated cytoplasm. These macrophages in lobomycosis, considered by some in the past to be granular myoblastoma cells, were rich in glycoproteins and appeared to be "Gaucher-like cells" in electronmicrographs. Further, a transition of macrophages containing organisms to Gaucher-like cells was noted. Antibodies to the fungus were demonstrated in the patient's serum by counter immunoelectrophoresis and indirect immunofluorescence studies. We postulate that the Gaucher-like cells formed because of the inability of host macrophages to digest glycoproteins in the capsule of this unusual fungus.
Skin lesions on an Atlantic bottlenosed dolphin, captured off the coast of Florida, were investigated and found to be histologically and microbiologically indistinguishable from those caused in humans by Loboa loboi. All attempts to isolate the etiologic agent or to transmit the infection to mice and monkeys ended in failure. Sight records of other suspected dolphin cases of lobomycosis in Florida waters are described along with citations of two previously confirmed and published dolphin infections.